Advancements in the understanding of gene expression and epidemiology combined with developments in technology have allowed for the correlation of genetic expression with, for example, disease states. An accurate correlation may enable risk assessment for an individual based on the expression profile of their individual cells. Further, drug screening and other research based protocols may quickly generate data in cell lines or tissue samples that can be extended to develop treatments for human disease.
Most of the methodologies available for evaluation of cell lines or tissue have well-known drawbacks. For example, most gene expression analyses extract RNA from solubilized tissue, which destroys cellular architecture such that expression profiles can not be associated with specific cell types. Analysis of small samples excludes the detection of a large number of potentially interesting low abundance gene products. Lack of standardization and reproducibility and amplification bias limit the interpretation of expression data resulting from methods such as RNA amplification and quantitative PCR (Bubendorf L., et al. J. Pathol. 195, 72 (2001); Capodieci P., et al. Diagn. Mol. Pathol. 7, 69 (1998); Femino, A. M., et al. Science 280, 585 (1998)). In addition, methods that require disaggregation of the sample, such as Southern, Northern, or Western blot analysis, are rendered less accurate by dilution of the malignant cells by the normal or otherwise non-malignant cells that are present in the same sample. Furthermore, the resulting loss of tissue architecture precludes the ability to correlate, for example, malignant cells with the presence of genetic abnormalities in a context that allows morphological specificity. This issue is particularly problematic in tissue types known to be heterogeneous, such as in human breast carcinoma, where a significant percentage of the cells present in any area may be non-malignant.
Another drawback is that many of the art recognized techniques require the tissue being analyzed to be fresh. Typically, however, it is not always possible in the clinical setting to work on cell lines or tissue as soon as they are available. Accordingly, cell lines or tissue are often preserved in paraffin. Processes for treating a paraffin-embedded tissue sample for gene analysis have been described, for example, U.S. Pat. Nos. 5,672,696 and 6,248,535. Typically treatments comprise treating tissue cells freed of paraffin with a solution containing a surfactant, a protease, etc. at room temperature to upwards of 60° C. for 4 to 48 hours to disrupt the tissue cells, removing impurities (i.e., substances other than nucleic acid) by a two-phase separation method (i.e., a method comprising separation into an aqueous phase containing the nucleic acid and an organic solvent phase containing denatured protein and the like by addition of one or more organic solvents such as phenol, chloroform, etc.), and then adding an alcohol to the residue to precipitate the nucleic acid in the aqueous phase (Jikken Igaku, Vol. 8, No. 9, pp. 84-88, 1990, Yodosha Co., Ltd.). While this technique allows for the analysis of gene expression, the purification disrupts cellular architecture and does not allow the application of in situ hybridization techniques.
As described in U.S. Pat. Nos. 5,750,340 or 6,165,723, in situ hybridization (ISH) is a powerful and versatile tool for the detection and localization of nucleic acids (DNA and RNA) within cell or tissue preparations. By the use of labeled DNA or RNA probes, the technique provides a high degree of spatial information in locating specific DNA or RNA target within individual cells or chromosomes. ISH is widely used for research and potentially for diagnosis in the areas of prenatal genetic disorders, and molecular cytogenetics. In the general area of molecular biology, ISH is used to detect gene expression, to map genes, to identify sites of gene expression, to localize target genes, and to identify and localize various viral and microbial infections. Currently, the application of the ISH technology research is being expanded into tumor diagnosis, preimplantation genetic diagnosis for in vitro fertilization, evaluation of bone marrow transplantation, and analysis of chromosome aneuploidy in interphase and metaphase nuclei.
In ISH, labeled nucleic acids (DNA or RNA) are hybridized to chromosomes, DNA or mRNAs in cells which are immobilized on microscope glass slides (In Situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In Situ Hybridization: In Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In Situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England (1992)). Numerous non-isotopic systems have been developed to visualize labeled DNA probes including, for example, a) fluorescence-based direct detection methods, b) the use of digoxigenin- and biotin-labeled DNA probes coupled with fluorescence detection methods, and c) the use of digoxigenin- and biotin-labeled DNA probes coupled with antibody-enzyme detection methods. When fluorescence-labeled nucleic acid (DNA or RNA) probes are hybridized to cellular DNA or RNA targets, the hybridized probes can be viewed directly using a fluorescence microscope. By using multiple nucleic acid probes with different fluorescence colors, simultaneous multicolored analysis (i.e., for multiple genes or RNAs) can be performed in a single step on a single target cell (Levsky et al. Science 2001). Fluorochrome-directly labeled nucleic acid probes eliminate the need for multi-layer detection procedures (e.g., antibody-based system), which allows for fast processing and also reduces non-specific background signals. Therefore, fluorescence in situ hybridization “FISH” has become an increasingly popular and valuable tool in both basic and clinical sciences.
Unfortunately, although FISH is an extremely useful technique, detection of mRNA, especially pre-mRNA (hnRNA, nascent RNA etc) in paraffin-embedded or otherwise fixed-treated cell lines or tissue (i.e., “fixed-treated tissue” defined as tissue that is not fresh frozen) is currently difficult, if not impossible. FISH is a highly sensitive assay that allows the detection of nucleic acid within undisturbed cellular and tissue architecture and the use of synthetic oligomer probes in FISH has improved the sensitivity of the process; however, to date FISH has only been successfully conducted in cells grown through cell-line culture. mRNA detection through FISH has not been successfully conducted in tissue until just recently (Nguyen et al., J Biol Chem, November 1; 277 (44):41960-9 (2002)); Paris et al., Science, July 13; 293 (5528):293-7 (2001)).
Detection is difficult for a number of reasons, including interference caused by the creation of chemical bonds during fixation processes as well as native autofluorescence in the cell lines or tissue. The ability to easily apply FISH to such cell lines or tissue would be of great interest because of the large amount of clinically relevant cell lines and tissue that have been (and continue to be) preserved in this fashion.
U.S. Pat. No. 5,856,089 describes in situ hybridization methods using nucleic acid probes for single copy sequences for detecting chromosomal structural abnormalities in fixed tissue obtained from a patient suspected of having a chromosomal structural abnormality. The methods include the use of bisulfite ion on the fixed cells.
U.S. Pat. No. 5,672,696 describes preparation of a sample for a gene analysis or high-purity nucleic acid suitable for gene amplification from a paraffin-embedded tissue sample comprising heating an aqueous suspension containing a surfactant having a protein-denaturation action and a deparaffinized tissue sample obtained from a paraffin-embedded tissue sample at 60° C. or higher. However, it is not an object of this patent to preserve the cellular architecture.
FISH has historically been combined with classical staining methodologies in an attempt to correlate genetic abnormalities with cellular morphology (see e.g., Anastasi et al., Blood 77:2456-2462 (1991); Anastasi et al., Blood 79:1796-1801 (1992); Anastasi et al., Blood 81:1580-1585 (1993); van Lom et al., Blood 82:884-888 (1992); Wolman et al., Diagnostic Molecular Pathology 1 (3): 192-199 (1992); Zitzelberger, Journal of Pathology 172:325-335 (1994)). However, several of these studies address hematological disorders where genetic changes are assessed in freshly fixed smears from bone marrow aspirates or peripheral blood specimens. U.S. Pat. No. 6,573,043 describes combining morphological staining and/or immunohistochemistry (IHC) with fluorescence in situ hybridization “FISH” within the same section of a tissue sample.
U.S. Pat. No. 6,534,266 describes an in situ hybridization method for detecting and specifically identifying transcription of a multiplicity of different target sequences in a cell. The method includes assigning a different bar code to at least five target sequences, with each target sequence containing at least one predetermined subsequence. Each bar code contains at least one fluorochrome, and at least one bar code comprises at least two different, spectrally distinguishable fluorochromes. A probe set specific for each target sequence is provided in the method. Each probe set contains a hybridization probe complementary to each subsequence in the target sequence. Each probe is labeled with a fluorochrome, and the fluorochromes in each probe set collectively correspond to the bar code for the target sequence of that probe set. Similar techniques are envisioned in combination with the invention disclosed herein.
Further, although spotted chip expression microarrays have been used extensively to detect the presence or absence of multiple specific mRNAs simultaneously in tissue, to date the effective application of this technique has been limited to fresh frozen tissue and does not describe an easy application utilizing paraffin-embedded or other fixed-treated tissue (for example, see United States Patent Publication Nos. 20030040035 and 20020192702). Because much of the cell lines and tissue available for scientific or medical study has been fixed, the ability to effectively use spotted chip arrays on fixed-treated cell lines and tissue would be of great potential value in (1) the discovery of the molecular mechanisms of the cell and its surrounding tissue in health and disease, (2) the creation of tests diagnostic of disease, (3) the creation of treatments therapeutic for disease, and (4) the identification of agents that are toxic to cells. Therefore, the present invention fulfills a need in the art by providing, for example, a process termed “mRNA liberation in fixed treated tissue or ‘MLIFFT’” to enable the detection of mRNA, especially pre-mRNA, in fixed treated tissue.
In this disclosure, we report the development of fluorescent in situ hybridization “FISH” protocols using cultured cells to yield expression profiles previously unobtainable by other methods (e.g., Levsky, J. M., et al. Science 297, 836 (2002); Kosman D., et al. Science, 305, 846 (2004)).